The present invention relates to novel human parathyroid hormone derivatives useful as hormone remedies and the production thereof.
Parathyroid hormone (hereinafter also briefly referred to as PTH) is a polypeptide hormone consisting of 84 amino acids which is secreted from the parathyroid, and one of the most important regulators for calcium metabolism. Accordingly, the application of human PTH to various bone diseases such as hypoparathyroidism and osteoporosis and further the application of human PTH antagonists to hypercalcemia and the like have been strongly desired.
The DNA sequence of human PTH was first revealed by G. N. Hendy et al. Proc. Natl. Acad. Sci. U.S.A., 78, 7365-7369 (1981)!. Since then, many attempts have been made to obtain human PTH by genetic engineering techniques. Recently, it has been expressed in amounts satisfiable from the industrial viewpoint with much effort for example, Wing L. Sung et al., J. Biol. Chem., 266, 2831-2835 (1991) and European Patent Unexamined Publication No. 483509!.
Human PTH has the following amino acid sequence: ##STR1## Its biological activity has previously been known to be reproducible by the fragment consisting of amino acid residues situated in the 1- to 34-positions on the N-terminal side (the positions of amino acid residues are hereinafter represented by the numbers corresponding to those of the sequence of human PTH (1-84) taking Ser as the 1-position) G. W. Tregear et al., Endocrinology, 93, 1349-1353 (1973)!, and many derivatives thereof have been synthesized. As to this (1-34) fragment, a peptide in which 2 to 6 amino acid residues on the N-terminal side are deleted is known to have PTH antagonist activity. Furthermore, the binding activity of a C-terminal portion, from the 35-position on, to a receptor L. G. Rao et al., Endocrinology, 117, 1632-1636 (1985)! and the activating action thereof to alkaline phosphatase T. M. Murray et al., Endocrinology, 124 1097-1099 (1989)! have recently been disclosed.
However, when natural type PTH (1-84) is actually used as a drug, it has some problems to be solved. For example, Met residues in a peptide chain are gradually oxidized even under ordinary conditions, and PTH whose Met residue is oxidized is significantly reduced in biological activity A. L. Frelinger III and J. E. Zull, J. Biol. Chem., 259, 5507-5513 (1984)!. Furthermore, it is generally preferred to use drugs as non-injection drugs from the simplicity and easiness of their administration. The modification of human PTH (1-84) is considered to make it possible to change the physicochemical properties of the drugs, for example, to allow the drugs to be easily absorbed from the mucous membrane.
It is the most common method to replace an amino acid(s) in a peptide chain by another amino acid(s) to attempt to improve the biological and physicochemical properties of a biologically active peptide, in order to solve such problems. Previously, the present inventors provided a high expression system of human PTH (1-84) in Escherichia coli (European Unexamined Patent Publication No. 483509), and succeeded in obtaining anti-oxidative derivatives by substituting other amino acid for Met residues of human PTH, utilizing this expression system. In addition, the present inventors obtained derivatives in which various amino acid residues in the center portion of the peptide chain are substituted by Cys residues. For example, a highly lipophilic group is specifically introduced into this SH group, or a dimer is formed through an S-S bond, whereby the derivative can be derived to an agonist having high affinity to a receptor or difficulty to undergo decomposition in vivo. Cyano-group can be introduced into the side-chain of the Cys residue followed by cleavage of the peptide bond to give an active fragment of PTH. Further, compounds in which several amino acid residues on the N-terminal side of PTH (1-34) are deleted are known to function as inhibitors N. Horiuchi et al., Science, 220, 1053-1055 (1983)!.